Fluorescent PCR (polymerase chain reaction) detection kit for vibrio cholerae and systematic identification method thereof

A detection kit and Vibrio cholerae technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganism measurement/inspection, etc., can solve the problems of enhanced toxicity, unstable results, long cycle, etc., and achieve saving detection The effect of reducing cost, shortening detection time, and improving detection rate

Inactive Publication Date: 2011-11-16
ZHEJIANG CENT FOR DISEASE CONTROL & PREVENTION
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional methods for detection of Vibrio cholerae mainly include morphology, biochemistry, coagulation test, etc. The cycle of these detections is long, generally more than 18 to 24 hours. strains) cannot be detected
Although some immunological methods are fast, the results are unstable, and the current routine detection methods only detect Vibrio cholerae of O1 and O139 serotypes, and cannot detect non-O1 and non-O139 serotype strains
At the same time, studies have clarified that virulence genes such as toxR and hlyA can be transferred from cholera O1 group toxin-producing strains to environmental non-O1 and O139 group strains, enhancing the toxicity of non-O1 and O139 group strains. Studies have shown that non-O1 and non-O139 group Group Vibrio cholerae is more suitable for survival and reproduction in seawater products than O1 and O139 group Vibrio cholerae, so it greatly threatens the food safety of seawater products

Method used

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  • Fluorescent PCR (polymerase chain reaction) detection kit for vibrio cholerae and systematic identification method thereof
  • Fluorescent PCR (polymerase chain reaction) detection kit for vibrio cholerae and systematic identification method thereof
  • Fluorescent PCR (polymerase chain reaction) detection kit for vibrio cholerae and systematic identification method thereof

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Experimental program
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Effect test

Embodiment 1

[0058] Embodiment 1: the acquisition of specific primers, fluorescent probes and standards

[0059] 1. Materials:

[0060] Bacterial genomic DNA extraction reagents were purchased from Treasure Bioengineering (Dalian) Co., Ltd.; pGEM-T-Easy cloning system and Taq DNA polymerase were purchased from Promega Company of the United States; sequencing reagents and 377 sequencer were all products of ABI Company of the United States. Spectrophotometer (Eppendorf Company), Bio-Rad icycler PCR instrument (Bio-Rad Company) and ABI7500FAST type quantitative PCR instrument (ABI Company)

[0061] 2. Primer and probe design and synthesis:

[0062] Using Vibrio cholerae outer membrane protein gene (ompW) (registration number DQ776044), toxin expression regulatory protein gene (toxR) (registration number HQ452873) and hemolysin gene (hlyA) (registration number GU230682) as templates, use Primer Express TM (V2.0, American ABI Company) software analyzes TaqMan primer and probe site, selects t...

Embodiment 2

[0086] Example 2: Systematic identification of Vibrio cholerae and its related toxin gene detection

[0087] 1. Identification of isolates from environmental samples:

[0088] Inoculate 30 bacterial strains to be identified on Columbia agar medium, culture in a 37°C incubator for 18-24 hours, pick a colorless, transparent, round, smooth and moist colony and puncture it on a three-sugar slant, culture it at 37°C for 18-24 hours Observe the results, and at the same time, the above-mentioned colonies are tested for oxidase, sticky silk and unsalted peptone water.

[0089] Santang slant: Colorless, transparent, round, smooth and moist colonies are punctured on the three sugar slant, cultured at 37°C for 18-24 hours, and the results are observed. The red and yellow on the slant are positive;

[0090] Sticky silk test: drop a large drop (about 15 μl) of the reagent on a glass slide or a plate, then take 1 loop of fresh agar culture of the tested bacteria, put it next to the reagent...

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Abstract

The invention provides a quick detection method and a fluorescent PCR (polymerase chain reaction) detection kit invented according to the typical biochemical characteristics of sucrose, arabinose, mannose, oxidase, haircuts, salt-free peptone water and the like of the vibrio cholerae. The invention designs primers and TaqMan probes according to the outer-membrane protein gene (ompW), the toxin expression regulation protein gene (toxR) and the hemolysin gene (hlyA), wherein the vibrio cholerae outer-membrane protein gene is used as a characteristic gene for identifying the vibrio cholerae, andthe toxin expression regulation protein gene and the hemolysin gene are used for discriminating toxin genes carried by the vibrio cholerae. The fluorescent detection method for detecting the vibrio cholerae and related toxin genes thereof is provided based on the three genes. According to the detection method, strains are primarily screened through biochemical reaction, the strains with positive results are confirmed by fluorescent quantitative PCR, and the strains with positive ompW genes are vibrio cholerae. The vibrio cholerae and related toxin genes thereof can be quickly, accurately and specifically detected by the biochemical reaction and fluorescent quantitative PCR.

Description

(1) Technical field [0001] The invention relates to a fluorescent PCR detection kit for three groups of Vibrio cholerae and a method for systematic identification of three groups of Vibrio cholerae using the kit. (2) Background technology [0002] Cholera is a kind of severe infectious disease caused by Vibrio cholerae with diarrhea as the main symptom. It is one of the ancient and widespread severe infectious diseases and has caused many pandemics in the world. Vibrio cholerae is the causative agent of cholera in humans. Vibrio cholerae has been divided into more than 200 serogroups according to the different bacterial (O) antigens, of which only O1 and O139 groups can cause cholera pandemics, and the rest of the serotypes are widely distributed in natural water bodies, generally not disease or only sporadic cases of diarrhea and intestinal infections. [0003] The Diarrhea Control Center of the World Health Organization divides Vibrio cholerae into 3 groups: O1 group Vib...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12R1/63
CPCY02A50/30
Inventor 罗芸叶菊莲张政金大智
Owner ZHEJIANG CENT FOR DISEASE CONTROL & PREVENTION
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